Process for detecting PrP using a macrocyclic adjuvant ligand

ABSTRACT

A process for detecting the forms of the prion pathogens responsible for subacute, transmissible, spongiform encephalopathies, including a macrocyclic adjuvant ligand (AML), free or bound to a support, that is added to a biological sample capable of containing PrP sc , the resulting suspension then being reacted with an anti-PrP sc  antibody, and the presence of PrP is then detected.

RELATED APPLICATION

This is a continuation of International Application No.PCT/FR2003/003857, with an international filing date of Dec. 19, 2003(WO 2004/059322 A1, published Jul. 15, 2004), which is based on FrenchPatent Application No. 02/16383, filed Dec. 20, 2002.

FIELD OF THE INVENTION

This invention relates to a process for detecting the forms of prionpathogens responsible for subacute, transmissible, spongiformencephalopathies.

BACKGROUND

The native or normal prion protein, designated PrP or PrP^(c), for thecellular prion protein is a glycoprotein broadly expressed in thelymphoid and neuronal cells of mammals.

Conformational changes of PrP^(c) result in the appearance and thepropagation of the protein pathogen PrP^(c), that is resistant to theproteinase K. This protein pathogen can be indifferently called PrP^(sc)or PrP^(res). Accumulation of PrP^(sc) in the organs of animals is atthe origin of numerous diseases and especially trembling. in smallruminants, of chronic cachetic disease (or chronic wasting disease“CWD”) of the elk and antelope, bovine spongiform encephalopathy (ESB)and Creutzfeld-Jakob disease. (MCJ) in humans.

The delayed appearance after an incubation period of 2 to 6 years andthe slow development of symptoms in cattle infected with ESB hasconsiderably slowed the development of epidemiological models. ESB istransmissible by ingestion to humans and has resulted in the appearanceof a new form of Creutzfeld-Jakob disease (vMJC).

Detecting the protein pathogen PrP^(sc) is difficult in infected animalsthat are otherwise healthy before the development of the disease andespecially in the blood and urine of diseased animals. It has beenestablished that PrP^(sc) present in animals intended for humanconsumption is transmitted to humans during the ingestion of infectedtissues. Thus, a major objective of public health is to avoid thistransmission by detecting the presence of PrP^(sc):

In animals intended for human consumption to remove them from the foodchain;

In blood donations and blood derivatives intended for transfusion tohumans. In fact, as the presence of the protein pathogen PrP^(sc) in theblood and the lymphoid liquids shows well before the cerebraldisturbance, and thus well before the possibility of detecting theneurological signs suggestive of a clinically declared prion disease,the physiopathology in humans is poorly recognized and, not being ableto carry out experimental infections as in sheep, the absence of adetection test in the blood or another biological fluids does not allowit to be studied and to thus prevent human to human transmission byblood donation or to treat infected persons before the cerebral lesionshave begun; and

In animal herds before the neurological stage, thus permitting theelimination of animals infected early before their arrival in theslaughterhouse.

Detecting the presence of PrP^(sc) in biological samples or animals hasthus become extremely important and several research teams aredeveloping methods of immunological detections (WO 02/086551). Moreover,methods of complexing peptides, small molecules or inhibitors toPrP^(sc) to treat vMJC constitute the subject of active research.However, those methods constantly come up against the difficulty ofidentifying PrP^(sc) in a reliable manner when it is in a low quantityin a biological sample and especially in biological fluids.

SUMMARY OF THE INVENTION

This invention relates to a process for detecting PrP including adding amacrocyclic adjuvant ligand (AML) that is free or linked to a support toa biological sample capable of containing PrP; reacting a resultingsuspension with an anti-PrP^(sc) antibody; and identifying the presenceof PrP.

This invention also relates to the process wherein the macrocyclicadjuvant ligand corresponds to the general formula (I) below:

in which R₁ represents a hydrogen atom, a hydroxyl group, an OR group oran OCOR group with R as defined below, R₂ represents a hydrogen atom, agroup R, COR, Pol, CH₂Pol in which Pol represents a phosphate, sulfate,amine, ammonium, carboxylic acid group and R is as defined below, R₃represents a hydrogen atom, a hydroxyl group, an OR group or an OCORgroup in which R is as defined below, R₄ represents a hydrogen atom, ahydroxyl group, an OR group, an OCH₂R group or an OCOR group in which Ris as defined below, Y is an atom of carbon, nitrogen or an atom ofsulfur, R₅ and R₆ are, each independently, absent or represent ahydrogen atom, a CH₂ group or R as defined below, or R₅ and R₆ togetherrepresent an atom of oxygen or of sulfur, X represents a CH₂ group or anatom of oxygen or of sulfur, m represents a positive integer equal to 0or 1, R represents a hydrogen atom or a hydrocarbon chain, saturated orunsaturated, branched or unbranched, cyclic or non-cyclic, substitutedor not substituted by a halogen group and carrying polar or non-polarfunctions, n is a positive integer between 3 and 15, and R₁ to R₅, R, X,Y and m can be different.

This invention further relates to the process wherein the macrocyclicadjuvant ligand corresponds to the general formula (Ia) below:

in which each R₂ group, taken independently, is a sulfate group or aphosphate group, R₇ is a (CH₂)_(r)—Z group in which Z is a COOEt, COOH,CN or NH₂ group, r is a positive integer between 1 and 20, p is apositive integer between 1 and 10, and n is a positive integer between 2and 10.

This invention still further relates to a macrocyclic adjuvant ligandgrafted onto a functionalized support in which the ligand corresponds tothe formula (Ib):

in which n is a positive integer between 4 and 8, each R₂ group, takenindependently, is a sulfate group or a phosphate group, R₈ represents a(CH₂)_(t)—(CO)_(s)—(NH₂) group or a (CH₂)_(t)—COOH group in which t is apositive integer between 0 and 6 and s is a positive integer between 0and 6, and the support is a solid support functionalized by an NHS groupor an NH₂ group and is a magnetic ball or a microplate.

This invention yet again relates to a diagnostic kit for diseasesinvolving PrP^(sc) including a macrocyclic adjuvant ligand of formula(1), free or bound to a support.

This invention further still relates to a kit for the immunologicaldosing of PrP^(sc) including a macrocyclic adjuvant ligand of formula(I), free or bound to a support.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparative example of the detection by immunoblotting ofPrP^(sc) on the one hand in a sample placed in the presence of aspecific macrocyclic adjuvant ligand (AML1) whose preparation isdescribed below and, on the other hand, in a sample not placed in thepresence of AML1.

FIG. 2 shows optical density curves of the detection of PrP^(sc) on theone hand in a sample placed in the presence of AML1 and on the otherhand in a sample not placed in the presence of AML1.

FIG. 3 shows images in scanning probe microscopy (SPM) in non-contactmode for dried films of the recombinant protein prion (recPrP) alone(A), of recPrP in the presence of ALM1 (13) and of AML1 alone (C).

FIG. 4 shows images in scanning probe microscopy (SPM) in non-contactmode for dried films of recPrP in the presence of AML in differentproportions: 1/1000 AML1/recPrP, 1/100 AML1/recPrP, 1/10 AML1/recPrP.

FIG. 5 shows a graphic representation giving the values of opticaldensity (DO) obtained as a function of the concentration of calyx-areneC6S grafted onto an activated amine plate (NIHS) after incubation of asolution of recombinant bovine PrP and disclosure by the anti-PrP AC23antibody marked with peroxidase.

FIG. 6 shows a graphic representation giving the values of opticaldensity (DO) obtained as a function of the concentration of calyx-areneC6S grafted on an activated amine ball (NHS) after incubation of asolution of human recombinant PrP dosed at 0.25 μg/ml by directdisclosure with the anti-PrP 8D11G12 antibody marked with peroxidase.

FIG. 7 shows a graphic representation giving the values of opticaldensity (DO) obtained after incubation with a dilution range of humanserum in PBST 0.05% placed in contact with calyx-arene C6S grafted on anactivated amine ball (NHS) by direct disclosure with the anti-PrP8D11G12 antibody marked with peroxidase.

FIG. 8 shows a graphic representation giving the values of opticaldensity (DO) obtained after placing calyx-arene C6S grafted on anactivated amine plate (NHS) in contact with samples of positive (LCR+)and negative (LCR−) human cerebrospinal fluid for MJC as a function ofthe heating by disclosure with the anti-PrP AC23 antibody marked withalkaline phosphatase.

FIG. 9 shows a graphic representation giving the values of opticaldensity (DO) obtained after placing calyx-arene C6S grafted on anactivated amine plate (NHS) in contact with PrP^(sc) extracted from thebrains of patients affected or not affected with Creutzfeld-Jakobdisease (MJC+/MJC−) by disclosure with the anti-PrP AC23 antibody markedwith alkaline phosphatase.

FIG. 10 shows a graphic representation giving the values of opticaldensity (DO) obtained as a function of the concentration of calyx-areneC6S grafted onto an activated amine plate (NHS) after being placed incontact with a sample of LCR of the patient not affected with MJC,diluted at ½ or ⅙ and in the absence of digestion by proteinase K.

FIG. 11 shows a graphic representation giving the values of opticaldensity (DO) obtained as a function of the concentration of calyx-areneC6S grafted onto an activated amine plate (NHS) after being placed incontact with a sample of LCR of the patients not affected with MJC(LCR−) or affected with this disease (LCR+), possibly diluted at ½(LCR+½ or LCR-½) and in the absence of digestion by proteinase K.

FIG. 12 shows a graphic representation giving the values of opticaldensity (DO) obtained as a function of the concentration of proteinase Kafter placing calyx-arene C6S grafted onto an activated amine plate(NHS) in contact with a sample of LCR of patients not affected with MJC(LCR−) or affected with this disease (LCR+).

DETAILED DESCRIPTION

This invention concerns a process permitting detecting the protein PrPand especially PrP^(sc) in dilutions in which it can not be detectedwith the methods currently used, which detection has had a 100%reliability in 119 samples that we tested. The process in accordancewith the invention advantageously permits the detection of PrP^(sc) tobe multiplied by a factor of 4.

More precisely, this invention concerns a process for detecting PrP and,in particular, PrP^(sc), characterized in that: a macrocyclic adjuvantligand (AML) that is free or linked to a support is added to abiological sample capable of containing PrP, the resulting suspension isthen reacted with an anti-PrP antibody, and the presence of PrP is thendetected.

The connection of the macrocyclic ligand to the support can be realizedin a known manner such as by adsorption or covalent bonding, covalentbonding, otherwise called grafting, being preferred.

When the attempt is made to detect PrP^(sc), the method can alsocomprise the supplementary step of treating the sample with proteinase Kbefore or after contact with the AML.

The presence of PrP can be realized by detecting the antibody reactionanti-PrP/PrP, which can be done by any means known in the art, andespecially by a sandwich method such as ELISA, immunoblotting, immunomicro-cantilever detection, mass spectrometry or by optical andspectroscopic analyses. Identification of the AML-PrP complex is made byprofilometric scanning techniques such as scanning reflectancemicroscopy, near-field scanning microscopy or confocal scanningmicroscopy.

The biological sample may come from an animal, including humans. Thesample preferably comes from the brain, tissues of the central nervoussystem or organs, in particular, the spleen or intestine. The sample maybe a biological fluid, especially cerebrospinal fluid or serum.

The term “macrocyclic ligand” denotes a compound that is capable ofbonding to PrP and, in particular, to PrP^(sc) and constituted of asuccession of cycles forming a macrocycle. Macrocyclic ligands areknown. It is possible to cite by way of non-limiting examplecyclophanes, metacyclophanes, cyclodextrins, cyclo(chromotropictetra-acids), spherands and cyclo[n]veratrylenes.

The macrocyclic adjuvant ligand is advantageously capable of bonding tosites that are contiguous or close in space, sites for bonding with anantibody and has the effect of increasing the antibody-PrP^(sc) bond.

The macrocyclic adjuvant is advantageously selected from the family ofmetacyclophanes. Among the metacyclophanes,thepara-sulfonato-calix-arenes functionalized on the phenolic face areparticularly advantageous. These compounds can be obtained in accordancewith the methodology described in Da Silva et al., J. Supramol. Chem. 1,(2001), pp. 135-138, the subject matter of which is hereby incorporatedby reference.

The macrocyclic adjuvant ligand advantageously corresponds to thegeneral formula (I) below:

in which

-   -   R₁ represents a hydrogen atom, a hydroxyl group, an OR group or        an OCOR group with R as defined below,    -   R₂ represents a hydrogen atom, a group R, COR, Pol, CH₂Pol in        which Pol represents a phosphate, sulfate, amine, ammonium,        carboxylic acid group and R is as defined below,    -   R₃ represents a hydrogen atom, a hydroxyl group, an OR group or        an OCOR group in which R is as defined below,    -   R₄ represents a hydrogen atom, a hydroxyl group, an OR group, an        OCH₂R group or an OCOR group in which R is as defined below,    -   Y is an atom of carbon, nitrogen or an atom of sulfur,    -   R₅ and R₆ are, each independently, absent or represent a        hydrogen atom, a CH₂ group or R as defined below, or    -   R₅ and R₆ together represent an atom of oxygen or of sulfur,    -   X represents a CH₂ group or an atom of oxygen or of sulfur,    -   m represents a positive integer equal to 0 or 1,    -   R represents a hydrogen atom or a hydrocarbon chain, saturated        or unsaturated, branched or unbranched, cyclic or non-cyclic,        substituted or not substituted by a halogen group and carrying        polar or non-polar functions,    -   n is a positive integer between 3 and 15, and    -   R₁ to R₅, R, X, Y and m can be different.

Thus, the compound of formula I is present in the form of a successionof n building blocks, characterized by the presence of a benzene ring,and in which the substituents of the ring can vary from one buildingblock to the other within the limit of their above definitions.

The macrocyclic ligands can be prepared according to known techniquesdescribed, e.g., in “Comprehensive Supramolecular Chemistry,” Pergamon,Oxford, 1996, the subject matter of which is hereby incorporated byreference.

The hydrocarbon chains, saturated or unsaturated, branched orunbranched, cyclic or non-cyclic, substituted or non-substituted by ahalogen group and carrying polar or non-polar functions are well-known.Representative examples include alkyls, alkenes, aryls, and saturatedcycles such as cyclohexane. An example of a non-polar group is CF₃ andexamples of polar groups are the substitutents Pol as defined above.

The macrocyclic adjuvant ligand may correspond to general formula Iabove and more precisely to general formula Ia below:

in which

-   -   each R₂ group, taken independently, is a sulfate group or a        phosphate group,    -   R₇ is a (CH₂)_(r)—Z group in which Z is a COOEt, COOH, CN or NH₂        group,    -   r is a positive integer between 1 and 20,    -   p is a positive integer between 1 and 10, and    -   n is a positive integer between 2 and 10.

The macrocyclic adjuvant ligand may be p-sulfonato-calix-[4]-arene,p-sulfonato-calix-≢6]-arene, p-sulfonato-calix-[8]-arene or one of theirderivatives.

The macrocyclic adjuvant ligand may also correspond to general formula Iabove and more precisely to general formula Ib below:

in which

-   -   n is a positive integer between 4 and 8,    -   each R₂ group, taken independently, is a sulfate group or a        phosphate group, and    -   R₈ represents a (CH₂)_(t)—(CO)_(S)—(NH₂) group or a        (CH₂)_(t)—COOH group in which t is a positive integer between 0        and 6 and s is a positive integer between 0 and 6.

The calyx-arene of formula Ib is advantageously such that each of thetwo R₂ groups is a sulfate group, n is 4, 6 or 8 and R₈ is a hydrogenatom, a CH₂COOH, a CH₂CONH₂ group or a CH₂CH₂NH₂ group.

The calyx-arene of formula Ib is preferably such that each of the two R₂groups is a sulfate group, n is a positive integer equal to 6 and R₇ isa (CH₂)₂—NH₂ group.

The ligand in accordance with the invention may be grafted onto a solidsupport. This support is functionalized by a function capable of forminga bond with a function carried by the ligand. The solid support may befunctionalized by an NHS (N-hydroxysuccinimide) bond or an NH₂ function.This function can react with a function carried on the ligand. In thatcase the calyx-arenes carrying a function capable of reacting to form abond with the functional bond of the solid support, in particular thecalyx-arenes carrying a NH₂ or COOH bond, are particularly preferred.

The invention also concerns a macrocyclic adjuvant ligand grafted onto afunctionalized support in which the ligand corresponds to formula Ib asdefined above in which n is a positive integer between 4 and 8 and thesupport is of the solid support type preferably functionalized by an NHSgroup or an NH₂ group and is preferably a magnetic ball or a microplate.Such a graft preserves the later interaction between the protein and theligand as well as the stereochemical presentation of the epitote fordetection by an anti-PrP antibody. The sites targeted by the graft onthe support are different than those that serve for the interaction withthe PrP.

The macrocyclic adjuvant ligand may be selected from the family ofcyclodextrins and, in particular, of cyclodextrins corresponding togeneral formula II:

in which

-   -   R₉ represents a hydrogen atom or a CH₂, OH, OR, OCOR, COR,        CH₂Pol, OCH₂R, SR, NR, Pol group and R is defined below,    -   R₁₀ and R₁ represent, each independently, a hydrogen atom or a        CH₂, OH, OR, OCOR, COR, CH₂Pol, OCH₂R, Pol group and R is        defined below,    -   Pol represents a phosphate, sulfate, amine, ammonium or        carboxylic acid group,    -   R represents a hydrocarbon chain, saturated or unsaturated,        branched or unbranched, cyclic or non-cyclic, substituted or        non-substituted by a halogen group and carrying polar or        non-polar functions,    -   n is a positive integer between 6 and 8, and    -   the substituents R₉ to R₁₁ can be different by nature in        accordance with the building blocks. Thus, the compound of        formula II is present in the form of a succession of n building        blocks characterized by the presence of a cyclic group, and the        substituents of this cyclic group can be variable from one        building block to the other within the limit of their above        definitions.

The macrocyclic adjuvant ligand may be selected from the family ofchromotropic cyclo-tetra-acids and, in particular, the chromotropiccyclo-tetra-acids corresponding to general formula III:

in which

-   -   R₁₂ and R₁₃ represent, each independently, a hydrogen atom or an        R or Pol group as defined below,    -   R₁₄ and R₁₅ represent, each independently, a hydrogen atom a CH₂        group or an R group as defined below, or    -   R₁₄ and R₁₅ together represent an atom of oxygen or of sulfur,    -   R₁₆ and R₁₇ represent, each independently, a hydrogen atom or a        hydroxyl group or an OR, OCH₂R, OCOR, SR, SCH₂R, SCOR group in        which R is defined as below,    -   M represents a hydrogen atom or an atom selected from Na, K, Li,        Cs, Rb, Mg and Ca,    -   Pol represents a phosphate, sulfate, amine, ammonium or        carboxylic acid group,    -   R represents a hydrocarbon chain, saturated or unsaturated,        branched or unbranched, cyclic or non-cyclic, substituted or        non-substituted by a halogen group and carrying polar or        non-polar functions,    -   n is a positive integer between 3 and 15, and    -   R₁₂ to R₁₇, M, Pol and R can be different by nature in        accordance with the building blocks. Thus, the compound of        formula III is present in the form of a succession of n building        blocks characterized by the presence of a cyclic group, and the        substituents of this cyclic group can be variable from one        building block to the other within the limit of their above        definitions.

The macrocyclic adjuvant ligand may also be selected from the family ofcyclo [n] veratrylenes, corresponding to general formula IV:

in which

-   -   R₁₈ represents a hydrogen atom or a CH₂, OH, OR, OCOR, COR,        CH₂Pol, OCH₂R, SR, NR, Pol group and R is as defined below,    -   R₁₉, R₂₀, R₂₁ and R₂₂ represent, each independently, a hydrogen        atom or a CH₂, OH, OR, OCOR, COR, CH₂Pol, OCH₂R, Pol group and R        is as defined below,        -   Pol represents a phosphate, sulfate, amine, ammonium or            carboxylic acid group, and        -   R represents a hydrocarbon chain, saturated or unsaturated,            branched or unbranched, cyclic or non-cyclic, substituted or            non-substituted by a halogen group and carrying polar or            non-polar functions, n is a positive integer between 1 and            10, and    -   R₁₈ to R₂₂, R, Pol and R can be different by nature in        accordance with the building blocks. Thus, the compound of        formula IV is present in the form of a succession of n building        blocks characterized by the presence of a cyclic group, and the        substituents of this cyclic group can be variable from one        building block to the other within the limit of their above        definitions.

Other subject matter of the invention comprises use of a macrocyclicadjuvant ligand (AML) for detecting the PrP^(sc) prion in a biologicalsample and, in particular, a ligand of formula I, Ia or Ib above, freeor bound to a support.

The invention also has as subject matter a kit for diagnosing diseasesof which PrP^(sc) is responsible and of the type ESB, trembling of smallruminants, Creutzfeld-Jakob, comprising the use of the macrocyclicadjuvant ligand and, in particular, a ligand of formula I, Ia or lbabove, free or bound to a support.

The invention also has as subject matter a kit for immunological dosingof PrP^(sc), comprising the use of a macrocyclic adjuvant ligand and, inparticular, a ligand of formula I, Ia or Ib above, free or bound to asupport.

Other advantages and features of the invention will appear from areading of the following examples concerning the amplification of thedetecting of PrP^(sc) when the biological sample is placed in thepresence of a free or grafted macrocyclic adjuvant ligand.

EXAMPLE 1 Preparation of Macrocyclic Adjuvant Ligands

1.1 Preparation of p-sulfonato-3,7-(2-carboxy-methyloxy)-calix-[6]-arene(called AML 1)

This macrocyclic adjuvant ligand AML1 has the general formula V:

This ligand was synthesized as follows: A suspension of calyx[6]arene inacetonitrile in the presence of a base equivalent (K₂CO₃) was agitatedon reflux. After 30 minutes, 0.5 equivalent of alkylating agent (ethylbromoacetate) was added to the reaction mixture, that was maintained onreflux and under agitation 24 hours. After cooling of the suspension thesolvent was evaporated under reduced pressure and the resulting solidwas dissolved in 200 ml CH₂Cl₂ and washed twice with a solution ofhydrochloric acid (1M) and once with demineralized water. After havingdried on magnesium sulfate (MgSO₄) the organic solvent was evaporatedunder reduced pressure and permits the obtention of a white powder. Thispowder was purified on a silica chromatography column (eluent: CH₂Cl₂:hexane; 4:1). The purified product was analyzed in RMN of ¹H, ¹³C and inmass spectrometry electrospray. The saponification of the carboxylicester was performed by potassium hydroxide in solution in a mixture ofwater:ethanol (30:70) under agitation at ambient temperature 24 hours.The precipitate formed was filtered, then washed with 50 ml hydrochloricacid (1M) and 50 ml water, then purified on a silica chromatographycolumn (eluent chloroform:methanol:acetic acid; 95:5:0.1%) and permitsthe obtention, after evaporation of the solvent, of a white andcrystalline solid. This product is analyzed in RMN of ¹H, ¹³C and inmass spectrometry electrospray. The sulfonation was performed by atreatment with sulfuric acid at 50° for 24 hours. The product wasprecipitated with ether and permits the obtention of AML1 in the form ofa white crystalline powder.

1.2 Preparation of p-sulfonato-3,7-(2-aminoethyloxy -calix-[6]-arene(called C6S)

This macrocyclic adjuvant ligand C6S has the general formula VI:

This ligand was prepared in accordance with the method described in EricDa Silva and Athony W. Coleman, Synthesis and Complexation Propertiestowards Amino Acids of Mono-Substituted p-sulfonato-calix-[n]-arene,Tetrahedron 59 (2003), pp. 7357-7364, the subject matter of which ishereby incorporated herein.

1.3 Grafting of Ligand C6S on Plates and on Balls

This ligand was coupled with a solid support (ball or plate) carrying anactive surface.

Grafting on plate:

The plates sNHS-activated plates” with 96 sinks came from the Covalabcompany (Lyon, France). 100 μl of a solution of ligand are dissolved atdifferent concentrations in phosphate buffer 50 mM, pH 8.2. The sinkswere washed 3 times (3×200 μl) MilliQ water after two hours ofincubation at 37° C. The plates were dried at ambient temperature beforebeing used.

Several plates were made at different concentrations in ligand C6S.These plates are defined as “plates C6S” in the entire text.

Grafting on balls:

4 ml of a solution of activated ball NHS (2×10⁹ balls/ml; Dynabeads®M270amine, Dynals company, Norway) were aliquoted in tubes of 1 ml. Theballs were centrifuged and precipitated by magnetization. Thesupernatant was removed and the balls washed 3 times with 1 ml water.The ball bottom was reworked with different volumes of solution ofcalixarene in a phosphate buffer 50 mM, pH 8.2. 600 ml, 120 ml, 60 mland 12 ml of a ligand solution at 50 mg/ml (phosphate buffer 50 mM, pH8.2) are added. The balls were agitated 24 hours at ambient temperature.The balls were washed 3 times with MilliQ water 18 Ω to eliminate theligand that did not react. The balls were preserved in 1 ml water toreconstitute the initial concentration of 2×10⁹ balls/ml. The ballsolution was then ready for use. These balls are defined as “balls C6S”in the entire text.

1.4 Grafting of AML1 onto a Modified Mineral Surface

1 ml 3-aminopropyltrimethoxysilane (ABCR company) were placed insolution in a solution of 50 ml (95% ethanol and 5% H420) for 5 min. Thesilicon plate was immersed into this solution for 4 min under agitation.The silicon plate was rinsed with ethanol, then the plate set to dry at130° C. for 15 min. 10 ml AML1 in solution 0.1 M in DMSO was coupled ona modified silicon plate using a coupling agent (DCC/HOBT). The samplewas dried 12 hours at 23° C.

EXAMPLE 2 Detection of PrP using AML1 not Coupled to a Solid Support

2.1 Preparation of Samples

The samples that were used for the examples of FIGS. 1 and 2 wereprepared as follows:

Sample without AML: 0.5 g brain tissue was crushed in 4.5 ml 5% glucosesolution to obtain a suspension with 10% weight/volume. 1 μg proteinaseK (Boehringer) in 10 μl was added per 100 μl brain homogenate at 10% inglucose at 5% (equivalent to 10 mg of brain). The solution was placedunder vortex and incubated at 37° C. for another hour. After theaddition of 100 μl denaturing Laemmli buffer, the mixture was heated 5minutes at 100° C., centrifuged at 12000 G for 5 mn, and thesupernatants recovered to make them migrate on SDS PAGE.

Sample with AML: 0.5 g brain tissue was crushed in 4.5 ml 5% glucosesolution to obtain a suspension with 10% weight/volume. 1.5 μgproteinase K (Boehringer) in 10 μl was added per 100 μl brain homogenateat 10% in glucose at 5% (equivalent to 10 mg of brain). The solution wasplaced under vortex and incubated at 37° C. for another hour. After theaddition of 100 μl denaturing Laemmli buffer, the mixture was heated 5minutes at 100° C., centrifuged at 12000 G for 5 mn, and thesupernatants recovered to make them migrate on SDS PAGE.

2.2 Process for Detection by Immunoblotting

After migration on a one-dimensional electrophoretic gel of 15%polyacrylic amide in the presence of sodium dodecyl sulfate (SDS PAGE)as described by Laemmli, Nature 227 (1970), pp. 680-685, the proteinswere transferred by electrophoresis onto nitrocellulose membranes andimmunoblotted at ambient temperature 60 minutes with a monoclonalantibody recognizing a specific epitope constituted of amino acids126-160. The secondary antibody ( 1/5000) was a goat antibody directedagainst the heavy and light chains of the mice immunoglobulinsconjugated on horseradish peroxidase (IgG H+L).

The blots were then washed and the signals detected by chemiluminescenceeither with an ECL kit (Amersham) on films (Biomex light, Kodak) or witha super Signal Ultra (Pierce) and visualization on Fluor S. Multimager(BioRad).

FIG. 1 is a comparative example that shows that detection of the prionis increased at least four times when the process of the invention isused over the detection of the same protein in the absence of AML1. Inthe absence of AML1, no PrP^(sc) is detected for dilutions greater than⅛^(th). In the presence of AML, detection of PrP^(sc) in the same sampleis possible up to a dilution of 1/32^(nd). The results of FIG. 2 showthe detection.

2.3 Microscopy Results

The samples used for the experiments in probe scanning microscopyreported in FIGS. 3 and 4 were prepared as follows:

Samples containing only macrocyclic adjuvant ligand: The samplescontaining macrocyclic adjuvant ligand (50 mM) were prepared bydepositing 10 μl AML solution and 10 μl water on freshly split mica andare dried 24 hours at 37° C.

Samples containing only recombinant PrP protein (recPrP): the samplescontaining recPrP were prepared by depositing 10 μl of solution of 1/10(v/v) AML1/recPrP, 1/100 (v/v) AML1/recPrP and 1/1000 (v/v) AML1/recPrPand 10 μl water on freshly cut mica and dried 24 hours at 37° C.

Samples containing macrocyclic adjuvant ligand and recPrP at the sametime: 1) Samples containing macrocyclic adjuvant ligand and recPrP atthe same time were prepared by depositing 1 μl AML solution and 1000 μlrecPrP on freshly split mica and dried for 24 hours at 37° C.; 2)Samples containing macrocyclic adjuvant ligand and recPrP at the sametime were prepared by depositing 1 μl AML solution and 100 μl recPrP onfreshly cut mica and dried for 24 hours at 37° C.; 3) Samples containingmacrocyclic adjuvant ligand and recPrP at the same time were prepared bydepositing 1 μl AML solution and 10 μl recPrP on freshly cut mica anddried for 24 hours at 37° C.

An imaging analysis is performed with a Thermomicroscope Explorer AFMequipped with a 100 μm tripod scanner in non-contact mode using elevatedresonance frequencies (F₀=320 kHz) of pyramidal cantilever with siliconeprobes at a scanning frequency of 1 Hz. The processing of the images isperformed with SPM1ab 5.1 software and presented non-filtered.

It follows from FIGS. 3 and 4 that only the films of recPrP show acharacteristic structure in circular aggregates. In the presence of AML,the structures observed for recPrP are modified sequentially, are anincreasing function of the quantity of AML and show building blocks thatare rounded and possibly orthogonal crystallite. The images of onlymacrocyclic adjuvant ligand (AML1) show structures that are similar, butsmaller than the building blocks of the recPrP-AML films.

Other experiments were carried out by atomic force microscopy.

A subsequent analysis by surface analysis of the rugosity is presentedin Table I. The use of these rugosity measurements can be extended totechniques of profilometric analyses. TABLE I AML/ AML/ AML/ AML recPrPrecPrP recPrP recPrP only 1/1000 1/100 1/10 only Ra (nm) 48  69, 6 123,9 134, 4  1, 11 RMS (nm)  58, 7  82, 8 149 163, 1  1, 95 Average height217, 7 233, 3 408, 7 469, 8 5, 4 Maximum height 448, 9 484, 3  894, 661056, 9  43, 17 value

$\begin{matrix}{R_{a} = {\frac{1}{N}{\sum\limits_{i = 1}^{N}{{z_{i} - \overset{\_}{z}}}}}} & \left( {{Formula}\quad A} \right) \\{{RMS} = {\sqrt{\frac{1}{N}}{\sum\limits_{i = 1}^{N}\left\langle {z_{i} - z} \right\rangle^{2}}}} & \left( {{Formula}\quad B} \right) \\{{AvgHeight} = {\frac{1}{N}{\sum\limits_{i = 1}^{N}z_{i}}}} & \left( {{Formula}\quad C} \right)\end{matrix}$

The calculated rugosity values are obtained with the ThermomicroscopeSPML 5.01 software. The average rugosity Ra is defined as the arithmeticmean of the deviations in height (Formula A), the root mean squarerugosity RMS is defined as the square root of the average value of thesquares of the distances of the points at an average image value(Formula B) and the average height of the sample (Formula C).

EXAMPLE 3 Detection of Recombinant PrP using the C6S Graft on a SolidSupport

3.1 Detection of Bovine Recombinant PrP on Plates

a) Protocol

The protocol used in this experiment was from a classic ELISA.

According to Example 1, grafting of activated amine plates (NHS) wasperformed for 6 hours with a range of sulfonated calix-6-arenes (C6S)diluted in a solution of PBS 50 mM (saline phosphate buffer). The plateswere then rinsed with distilled water before being saturated with asolution of PBST (PBS Tween) (0.05% milk 5%) for 1 hour at ambienttemperature. After rinsing, a solution of recombinant bovine proteinprion (PrP) diluted to a concentration of 0.25 μg/ml was deposited inPBST 0.05%. It was incubated one hour at ambient temperature. The platewas rinsed again three times. It was then incubated with a solution ofanti-PrP antibodies marked with peroxidase diluted at 0.5 μg/ml in PBST0.05% for one hour at ambient temperature. The antibody used recognizesthe region defined by the amino acids 145-154 of human PrP and thehomologous regions of animal PrP (AC23). Finally, after a new rinsingcycle the developer, a solution of OPD (ortho-phenylene diamine), wasadded and incubated 10 minutes at ambient temperature away from light.The reaction was stopped with the aid of a solution of H₂SO₄. The signalobtained was read with the aid of a spectrophotometer at 495 nm.

b) Results

The results are indicated in FIG. 5, that shows that the optical density(DO) measured in the ordinate reflects the quantity of PrP captured bythe C6C'es and remaining caught after washings. The signal obtained forthe different graftings shows that the recombinant bovine PrP iscaptured well by the C6S'es. The bovine recombinant PrP fixes to theC6S'es grafted on the NHS plates.

3.2 Detection of Recombinant Human PrP on Balls

a) Experiment conditions

According to Example 1, the NHS balls were first placed in contact witha solution of C6S diluted in distilled water for grafting the C6S ontothe balls. After this period, the balls were washed with distilled waterthen with PBST 0.05%. A solution of recombinant human PrP dosed at 0.25μg/ml in PBST 0.05% is prepared in parallel. Then, a range of C6S ballswas realized by adding: 0, 0.1, 1, 5 and 10 μl of the ball solution per100 μl of the PrP solution. The incubation lasts 1 hour at 37° C., thenthe balls were separated from the supernatant by magnetization. The PrPfixed on the balls was dosed directly with the aid of an antibody markedin accordance with a sandwich method close to the ELISA method.

The developer antibody marked with peroxidase is incubated one hour at37° after rinsings of the balls by a solution of PBST 0.05%. The ballsare separated from the supernatant by magnetization before being washedagain, then revealed by a solution of OPD incubated 10 minutes. Thereaction was stopped with the aid of a solution of H₂SO₄. The developerantibody used was 8D11G12 (bioMerieux, France).

The reading of the signal obtained was made with the aid of a 495 nmspectrophotometer.

b) Results

The results of the developments on ball are indicated in FIG. 6 and showthat the C6S'es are correctly grafted without loss of function on theNHS supports, but especially retain their property of capturing therecombinant PrP of different species and even amplify the signalobtained with the specific antibodies of this protein in a reproduciblemanner.

EXAMPLE 4 Detection of Physiological PrP using C6S Grafted on a SolidSupport

4.1 Human Serum on Balls

a) Protocol

According to Example 1, the NHS balls were first placed in contact witha dilute solution of C6S in distilled water for grafting. After thisperiod, the balls were washed with distilled water then with 0.05% PBST.

A serum range was prepared by dilution in PBS. A solution of humanrecombinant PrP dosed at 0.25 μg/ml into PBS was prepared in parallel aswell as a solution of 0.05% PBST milk 5% that served as positive andnegative controls.

The balls were added at the rate of 5 μl of a solution of 2.10^(e)9balls/ml per 250 μl of sample. The incubation took 1 hour at 37° C.under agitation. The balls were then separated from the supernatant bymagnetization. The PrP fixed on the balls was dosed directly with theaid of a marked antibody.

The developer on the balls used a protocol close to ELISA. The developerantibody marked with peroxidase was incubated for one hour at 37° C. andunder gentle agitation after rinsing of the balls by a solution of 0.05%PBST. The balls were separated from the supernatant by magnetizationbefore being rewashed then revealed by a solution of OPD incubated for10 minutes. The reaction was stopped with the aid of a solution ofH₂SO₄. The developer antibody used was 8D11G12.

The reading of the signal obtained was made with the aid of a 495 nmspectrophotometer.

b) Results

The results are indicated in FIG. 7, that shows that:

The negative control in the milk permits evaluation of the backgroundnoise, whereas the positive control in a solution of 0.25 μg/ml PrPpermits a verification that the experiment functioned and isinterpretable.

The C6S'es grafted on the NHS balls captured the physiological PrPpresent in human serum. Moreover, the capture was better when thedilution of the serum was augmented with a saturation plate achieved forthe dilutions at ⅕^(th) and 1/10^(th).

c) Conclusion

Surprisingly, the C6S'es grafted on NHS balls captured the physiologicalPrP present in human serum. The dilutions of the serum permit the signalobtained on the balls to be augmented.

4.2 Liquid Human Cerebrospinal Fluid (LCR) on Plates

a) Experiment Conditions

The samples of LCR were first divided in tubes of equivalent volumes.Two dilution points: pure and 12 are established for each sample. Then,they were subjected to three types of heat treatment: 30 minutes at 56°C., 15 minutes at 75° C. or 5 minutes at 95° C. A control withoutheating was performed in parallel for the pure samples.

According to Example 1, in parallel, the NHS plates were placed incontact for 6 hours with a solution of C6S diluted in a solution of PBS50 mM for grafting.

The plates were then rinsed with distilled water then with 0.05% PBST.

The samples were then deposited and incubated one night at 2-8° C. Theplate was then rinsed six times. It was then incubated with a solutionof anti-PrP antibody marked with biotin diluted at 0.5 μg/ml in 0.05%PBST for one hour at ambient temperature. The antibody used is AC23.

After a new rinsing cycle, a solution of streptavidine-PAL (alkalinephosphatase) was added and incubated for 20 minutes at ambienttemperature. The plate was rinsed a last time and the developer solutionof PNPP deposited and incubated for 30 minutes at 37° C. The reactionwas stopped with the aid of a solution of NaOH 0.4N.

The reading of the signal obtained was made with the aid of a 495 nmspectrophotometer against a 490 nm filter.

b) Results

The results are indicated in FIG. 8, that shows that the physiologicalPrP can be dosed in the LCR.

EXAMPLE 5 Detection of PrP^(sc) in the Brain using C6S Grafted onto aSolid Support

5.1 Detection on Plate

a) Experiment Conditions

The first stage consists of obtaining PrP^(sc) purified by extractionfrom the brains of patients affected or not affected byCreutzfeldt-Jakob disease (MJC). A sample of 260 mg of each brain wastaken, then crushed to obtain a 10% homogenate in 5% glucose. The groundmatter was then filtered with the aid of a needle. A digestion stage byproteinase K followed. It was used at a concentration of 20 pg per 100mg of tissue at 37° C. for one hour. Then, 650 μl of a 30% Sarkosylsolution was added. A cushion of 400 μL saccharose was deposited inparallel on the bottom of a tube for ultracentrifugation. The sample wasthen deposited on this cushion. The tubes were completed before beingwelded, then ultracentrifuged for 2 hours at 20° C. at 100,000 rpm. Thesupernatants were eliminated and the tube walls roughly dried withabsorbent paper. The bottoms were then put in tris maleate. The sampleswere then heated for five minutes at 95° then centrifuged 15 minutes at12,000 rpm at 20° C. The samples were preserved at −80° C. until theirnext use. The success of this first stage was confirmed by Western blot.

b) Test on calix-6-sulfonates

According to Example 1, grafting of the NHS plates was performed for 6hours with a range of C6S diluted in a solution of 50 mM PBST. They werefirst saturated with a solution of 0.05% PBST milk 5% for 6 hours atambient temperature. They were then washed three times with a solutionof 0.05% PBST. The sample previously diluted in tris maleate wasdeposited on the plate and incubated one night at 2-8° C. The plate wasthen washed 6 times in 0.05% PBST. The solution of developer antibodywas incubated for 1 hour at ambient temperature. After a new series ofwashing, the streptavidine-PAL was incubated for 20 minutes. The platewas washed a last time before the deposit of PNPP (paranitrophenylphosphate), that was incubated for 30 minutes at 37° C. The developerreaction was then stopped by the addition of soda.

Reading the DO was performed at 405 nm against a filter with 490 nm withthe aid of a spectrophotometer.

c) Results

The results are indicated in FIG. 9, that shows that the PrP^(sc) of thesamples from patients stricken with Creutzfeld-Jakob disease issurprisingly detectable by the process of the invention using a captureby C6S'es grafted on NHS plates and detection by an antibody directedspecifically against the protein PrP, which is validated by the positivePrP recombinant human control and negative control PBST 0.05%.

EXAMPLE 6 Detection of PrP^(sc) in LCR and using C6S Grafted on a SolidSupport

6.1 Preparation of Samples

The specimens were constituted of samples of cerebrospinal fluid (LCR)that were preferably non-hemolyzed and without cellular debris. Thesamples were anonymous and preserved and frozen at (−80° C.) in tubeswith walls that were slightly absorptive for proteins (“pre-lubricatedmicro centrifuge tube 1.7 ml”, Marsh Biomedical Products, ref. T6050G).

They were divided into two categories:

Samples positive for MJC.

These were postmortem LCR recovered during the autopsy by puncture inthe cisterns and of which the research of the PrP^(sc) in the cerebraltissue by Western blot permitted the confirmation of the diagnosis ofMJC (certain cases). During autopsy, the LCR taken was immediatelydistributed into the tubes previously described without previouscentrifugation (with the exception of samples having a significantamount of cellular debris) and the aliquots frozen at −80° C. Samplesnegative for MCJ. They are of 2 types:

Postmortem LCR recovered during the autopsy and of which the research ofthe PrP^(sc) in the cerebral tissue by Western blot permitted theelimination of the diagnosis of MJC (controls negative for MJC).

LCR of patients not afflicted with MJJ and stemming from externalventricular derivations (DVE). The storing conditions of the sampleswere identical to those previously described.

6.2 ELISA Approach

a) Analytical Principle

The samples were simply incubated at an elevated temperature for a giventime, then, after cooling, diluted to ½ or ⅕ in a buffer compatible withan immunometric analysis (ELISA) ( tris maleate buffer pH 8). When thedigestion by proteinase K occurred before depositing on plate C6S, thesamples were digested by proteinase K for 15 minutes and then depositedon plate C6S without previous inhibition.

The term ELISA is employed even though the capture is not immunologicalbecause a reaction of the type ligand/refining agent with immunologicaldevelopment is concerned.

The ELISA techniques are immuno-enzymological quantitative techniquespermitting the dosing of the investigated antigen by the transformationof a substrate into a soluble, measurable product proportional to thequantity of antigen present in the sample. The technique used herecomprised the same steps as a non-competitive classical ELISA sandwichwith the exception of the sensitizing of the cupules. In fact, theanti-prion capture antibody was replaced by the sulfonatedcalyx-6-arenes grafted on the NHS groups of the microplates by theiramine function.

After immobilization of the C6S'es on the bottom of the cupules of amicroplate and saturation of the non-specific sites for the fixation ofthe antigen, the sample was deposited and the plate incubated to permitbonding of the antigen to the C6S. Then, after several washings, thedeveloper antibody specific for the prion protein and marked was added.The complexes formed were visualized by colorimetric method after theaddition of the enzyme substrate, that is transformed into a coloredproduct whose absorbency was determined with the aid of a microplatereader (automated spectrophotometer).

b) Mode of Operation

A volume of LCR was incubated in a dry stainless steel water pan at 75°C. for 15 minutes. After cooling, the sample was diluted 5 times in trismaleate buffer 0.2 M, pH 8 (addition of 4 volumes of buffer) and eitherdeposited directly on a sensitized microplate in advance by the C6S'esor digested by proteinase K for 15 minutes at 37°, then deposited onmicroplate.

The successive stages of the immunometric analysis are enumerated belowin chronological order:

-   -   1. Grafting the sulfonated calyx-6-arenes C6S'es on the        activated amine plates (NHS) was performed in accordance with        the method mentioned above for 6 hours at ambient temperature in        accordance with a concentration range or in accordance with a        fixed concentration, diluted in a solution of PBS 50 mM.    -   2. The plates were then washed 3 times in PBST 0.05%, then        saturated in PBST 0.05% milk 5% for 1 hour at 37° C.    -   3. After washing, 100 ml LCR prepared in accordance with the        analytical principle described above were deposited by sinks;        the plates were incubated for 1:30 h at 37° C.    -   4. After washing, 100 ml of developer antibody was deposited by        sinks (AC23-biotin at 0.5 μg/ml). The plates were incubated 1 h        at ambient temperature.    -   5. After washing, the complexes formed are disclosed:        -   5.1 If the disclosure antibody was coupled to biotin, 100 ml            of a solution of SA-PAL diluted to 1/20000 in PBST buffer            0.05% was deposited per sink. The plates were incubated 20            minutes at ambient temperature. After washing, 100 ml PNPP            was deposited by sinks. The plates were incubated IO to 30            minutes at 37° C. At the end of the incubation, the            colorimetric reaction was stopped by addition of 50 ml NaOH            at 0.4 N into each sink. The optical density was then            measured by spectrophotometer with 405 nm against a filter            with 450 nm.        -   5.2 If the disclosure antibody was coupled to peroxidase,            100 ml OPD was deposited by sinks. The plates were incubated            10 minutes in the dark and at ambient temperature.

At the end of the incubation, the colorimetric reaction was stopped byaddition of 50 ml H₂SO₄ at 1.8 N into each sink. The optical density wasthen measured with a spectrophotometer with 495 nm.

b) Results

-   -   1. Verification of the association of sulfonated monoamines        calyx-6-arenes (C6S grafted onto microplate with the protein        prion of cerebrospinal fluid (LCR):

A cerebrospinal fluid of the patient not afflicted with Creutzfeld-Jakobdisease was heated 15 minutes and 75° C. and then diluted to one half or⅙^(th) in a buffer compatible with an immunological disclosure in ELISA.The samples were not digested by proteinase K and thus deposited on aplate grafted with C6S'es in accordance with a concentration rangecomprised between 1.8 and 18 μg/ml. After incubation, the immunologicaldisclosure of the antigen captured on the C6S'es was ensured by theantibody AC23 coupled with peroxidase, used at 0.5 μg/ml. The resultsare shown in FIG. 10.

These results show that the C6S'es enter readily into a combination witha form of protein prion in the LCR since a signal was observed from thefirst concentration of C6S'es immobilized at the bottom of the cupules.

-   -   2. Verification of the association of the C6S'es with the        pathological protein prion in heterogeneous phase without using        proteinase K:

An LCR of a patient not afflicted with MJJ (LCR−) and an LCR of apatient deceased from MCJ (MJC+) non-diluted or diluted to 1/2 undergodifferent types of heat treatment. They are then deposited on a plategrafted with the C6S'es at 7.2 μg/ml. After washing, the immunologicaldisclosure was ensured by the antibody anti-prion AC23 coupled withbiotin.

The results are shown graphically in FIG. 11.

These results show an adsorption of the protein prion on the C6S'esgrafted on the plate in the absence of treatment by proteinase K, moreefficacious for the positive sample than for the negative sample. Thedifferent heat treatments slightly influence the efficacy of theadsorption of the protein prion on the C6S'es in the absence of dilutionand, furthermore, when the sample is diluted. Thus, the test conditionsfavoring the adsorption of the protein prion on the MN C6S'es inheterogeneous phase appear to be a dilution of the samples and atreatment for 5 minutes at 95° C.

-   -   3. Verification of the association of the C6S'es with the        pathological protein prion in heterogeneous phase with the using        of proteinase K:

A stage of digestion by proteinase K was used for 15 minutes at 37° C.in pre-analytic treatment before capture on plate C6C on the digestingmixture for 1 hour at 37° C. The elimination of the residual proteinaseK was ensured by successive washings in PBS Tween buffer 0.05%.

The results are indicated in FIG. 12.

These results show a difference of optical density in favor of thepositive sample for MJC after digestion by proteinase K. Thedifferential is visible from the first concentration of proteinase Ktested (0.5 μg/ml).

d) Conclusion

Detection of the protein prion in the LCR is made possible in accordancewith the invention. In fact, in the absence of digestion by proteinase Kthe detection of the total PrP (cellular and pathological) is amplifiedin the presence of the sulfonated calyx-6-arenes and, in an unexpectedmanner, the PrP^(sc) is preferentially detected. The use of these C6S'esin capture after digestion of the samples by proteinase K permits thedetection of the PrP^(sc) of the LCR by furnishing a signalsignificantly different between the LCR stemming from patients notafflicted with MJC and the LCR stemming from patients affected with MJC.

1-9. (canceled)
 10. The process according to claim 9, wherein themacrocyclic adjuvant ligand corresponds to the general formula (Ia)below:

in which each R₂ group, taken independently, is a sulfate group or aphosphate group, R₇ is a (CH₂)r-Z group in which Z is a COOEt, COOH, CNor NH₂ group, r is a positive integer between 1 and 20, p is a positiveinteger between 1 and 10, and n is a positive integer between 2 and 10.11. The process according to claim 9, wherein the ligand corresponds toformula (Ib) below:

in which n is a positive integer between 4 and 8, each R₂ group, takenindependently, is a sulfate group or a phosphate group, and R₈represents a (CH₂)_(t)—(CO)_(s)—(NH₂) group or a (CH₂)_(t)—COOH group inwhich t is a positive integer between 0 and 6 and s is a positiveinteger between 0 and
 6. 12. The process according to claim 11, whereinthe ligand is a calyx-arene of formula (Ib) in which each of the twogroups R₂ is a sulfate group, n is 4, 6 or 8 and R₈ is a hydrogen atom,a CH₂COOH, group, a CH₂CONH₂ group or a CH₂CH₂NH₂ group.
 13. The processaccording to claim 11, wherein each of the two R₂ groups is a sulfategroup, n is a positive integer equal to 6 and R₇ is a (CH₂)₂-NH₂ group.14. The process according to claim 7, wherein calyx arene is bound to afunctionalized support by an NHS or NH₂ function.
 15. A macrocyclicadjuvant ligand grafted onto a functionalized support in which theligand corresponds to the formula (Ib):

in which n is a positive integer between 4 and 8, each R₂ group, takenindependently, is a sulfate group or a phosphate group, R₈ represents a(CH₂)_(t)—(CO)_(s)—(NH₂) group or a (CH₂)_(t)—COOH group in which t is apositive integer between 0 and 6 and s is a positive integer between 0and 6, and the support is a solid support functionalized by an NHS groupor an NH₂ group and is a magnetic ball or a microplate.
 16. A diagnostickit for diseases involving PrP^(sc) comprising a macrocyclic adjuvantligand of formula (I), as defined in claim 9, free or bound to asupport.
 17. A diagnostic kit for diseases involving PrP^(sc) comprisinga macrocyclic adjuvant ligand of formula (Ia), as defined in claim 10,free or bound to a support.
 18. A diagnostic kit for diseases involvingPrP^(sc) comprising a macrocyclic adjuvant ligand of formula (Ib), asdefined in claim 15, free or bound to a support.
 19. A kit for theimmunological dosing of PrP^(sc) comprising a macrocyclic adjuvantligand of formula (I), as defined in claim 9, free or bound to asupport.
 20. A kit for the immunological dosing of PrP^(sc) comprising amacrocyclic adjuvant ligand of formula (Ia), as defined in claim 10,free or bound to a support.
 21. A kit for the immunological dosing ofPrP^(sc) comprising a macrocyclic adjuvant ligand of formula (Ib), asdefined in claim 15, free or bound to a support.
 22. A process fordetecting PrP^(sc), comprising: adding a macrocyclic adjuvant ligand(AML) that is free or linked to a support to a biological sample thatmay contain PrP^(sc) to form a suspension; reacting the suspension withan anti-PrP antibody subjecting the biological sample to conditions thatdegrade PrP, either before or after reacting adding the macrocyclicadjuvant ligand (AML); and identifying the presence of PrP^(sc); whereinsaid AML is selected from the group consisting of metacyclophanes,cyclodextrins and veratrylenes.